Skin blood flow (SKBF) demonstrates a biphasic response to innocuous, local heating. Much about the mechanism of the first phase is unknown. A type of ion channel (TRPV3) sensitive to and increasingly activated by temperatures from ~33 to ~45°C may be involved. TRPV3 channels are abundantly located in the keratinocytes and are believed to elicit the release of ATP, a putative cutaneous vasodilator, upon activation. This study investigated the possibility that TRPV3 channels and ATP have a role in the first phase of the SBKF response to local heat. Fifteen young, healthy subjects participated in the study. Two microdialysis probes were inserted into the dermis on the forearm. Using a peltier module, the skin above the probes (3cm x 3cm) was heated to 31, 35, 39, and 43°C to manipulate the level of activation of TRPV3 channels for eight minutes each. The probes were perfused with 0.9% saline at 2µl/min. Dialysate from each phase was analyzed for the concentration of ATP ([ATP]d). Cutaneous vascular conductance (CVC), measured by laser Doppler flowmetry, was monitored throughout. The [ATP]d decreased significantly when the skin was heated to temperatures known to strongly activate TRPV3 channels (i.e 39 and 43°C). [ATP]d demonstrated no relationship with CVC and only a very weak relationship with peltier temperature (r2 = 0.02, p<0.05). These data indicate that local heating and presumably heat-induced activation of the TRPV3 channels results in the decrease, not increase, of the release of ATP in human skin, and that the [ATP]d is not related to changes in skin blood flow. Significant dilation was observed at 35°C. This threshold, which is several degrees lower than the threshold previously reported, suggests that the TRPV3 channels may be involved in the dilator response in some way independent of interstitial ATP.

Neural circuitry, or how neurons connect across brain regions to form functional units, is the fundamental basis of all brain processing and behavior. There are several neural circuit analysis tools available across different model organisms, but currently the field lacks a comprehensive method that can 1) target post-synaptic neurons using a pre-synaptic driver line, 2) assess post-synaptic neuron morphology, and 3) test behavioral response of the post-synaptic neurons in an isolated manner. This work will present FLIPSOT, or Functional Labeling of Individualized Post-Synaptic Neurons using Optogenetics and trans-Tango, which is a method developed to fulfill all three of these conditions. FLIPSOT uses a pre-synaptic driver line to drive trans-Tango, triggering heat-shock-dependent expression of post-synaptic optogenetic receptors. When heat shocked for a suitable duration of time, optogenetic activation or inhibition is made possible in a randomized selection of post-synaptic cells, allowing testing and comparison of function. Finally, imaging of each brain confirms which neurons were targeted per animal, and analysis across trials can reveal which post-synaptic neurons are necessary and/or sufficient for the relevant behavior. FLIPSOT is then tested within Drosophila melanogaster to evaluate the necessity and sufficiency of post-synaptic neurons in the Drosophila Heating Cell circuit, which is a circuit that functions to drive warmth avoidance behavior. FLIPSOT presents a new combinatory tool for evaluation of behavioral necessity and sufficiency of post-synaptic cells. The tool can easily be utilized to test many different behaviors and circuits through modification of the pre-synaptic driver line. Lastly, the success of this tool within flies paves the way for possible future adaptation in other model organisms, including mammals. / Doctor of Philosophy / The human brain is made up of billions of neurons, each of which are interconnected in various ways to allow communication. When a group of connected neurons work together to carry out a specific function, that group is known as a neural circuit. Neural circuits are the physical basis of brain activity, and different circuits are necessary for all bodily functions, including breathing, movement, regulation of sleep, memory, and all senses. Disruptions in neural circuits can be found in many brain-related diseases and disorders such as depression, anxiety, and Alzheimer's disease. One example of a neural circuit is that of temperature sensation. When someone holds a cube of ice, temperature-sensing neurons in the hand pass signals along neurons in the spine until they reach the brain. There, the signals are carried to various brain regions to be processed and recognized as cold, and eventually, pain. When the sensory signals of cold and pain grow too prominent to ignore, the person may move to avoid the feeling. In this case, the brain will send signals back down to neurons responsible for movement in the arm, allowing the person to drop the ice cube. Avoidance of temperatures that are too warm or cold is an evolutionary trait that is important in preventing the body from harm. Even in a relatively simple system like temperature sensation, neural circuits can be complex and difficult to study, especially in higher order organisms such as mammals. For this reason, it can be beneficial to use simpler animals such as Drosophila melanogaster, or the common fruit fly. Flies have far fewer neurons than humans, meaning their neuronal connections are also significantly less complicated, and there are many genetic tools available in flies that aren't available in mammalian models such as mice. Additionally, flies are inexpensive, easy to raise, and grow quickly, making them ideal for troubleshooting new tools and replicating experiments. Though somewhat different in anatomy, fly brain function is similar enough to humans and other mammals that findings can often be applied across species. Studies in flies can also be applied in other insects, such as mosquitoes, which are notorious for carrying deadly diseases. Though there are several available tools in flies to study neural circuits, many tools are better for usage in sensory neurons themselves than in the neurons that carry signals in the brain afterward. This work presents a new tool, abbreviated as FLIPSOT, that modifies and combines several existing genetic methods in order to help examine those higher order neurons. FLIPSOT allows users to determine which higher order neurons are important in leading to behavioral responses, as opposed to carrying the signal to other brain regions, such as those associated with memory. Then, FLIPSOT is implemented in a warmth-sensing neural circuit known as the Heating Cell (HC) circuit and used to identify the higher order neurons needed for fly warmth avoidance. Development of tools such as FLIPSOT helps to expand our knowledge in the fields of neural circuits and behavior. Genetic tools can also be more easily tested in flies prior to attempting to implement them in other organisms, such as mice. Finally, studying temperature in flies can help create a deeper understanding of how temperature sensation works in all animals, including humans.

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The ability to sense humidity and wetness is an important sensory attribute for many species across the animal kingdom, including humans. Although this sensory ability plays an important role in many human physiological and behavioural functions, as humans largest sensory organ i.e. the skin seems not to be provided with specific receptors for the sensation of wetness (i.e. hygroreceptors), the neurophysiological mechanisms underlying this complex sensory experience are still poorly understood. The aim of this Thesis was to investigate the neurophysiological mechanisms underpinning humans remarkable ability to sense skin wetness despite the lack of specific skin hygroreceptors. It was hypothesised that humans could learn to perceive the wetness experienced when the skin is in contact with a wet surface or when sweat is produced through a complex multisensory integration of thermal (i.e. heat transfer) and tactile (i.e. mechanical pressure and friction) inputs generated by the interaction between skin, moisture and (if donned) clothing. Hence, as both thermal and tactile skin afferents could contribute significantly to drive the perception of skin wetness, their role in the peripheral and central sensory integration of skin wetness perception was investigated, both under conditions of skin s contact with an external (dry or wet) stimulus as well as during the active production of sweat. A series of experimental studies were performed, aiming to isolate the contribution of each sensory cue (i.e. thermal and tactile) to the perception of skin wetness during rest and exercise, as well as under different environmental conditions. It was found that it is not the contact of the skin with moisture per se, but rather the integration of particular sensory inputs which drives the perception of skin wetness during both the contact with an external (dry or wet) surface, as well as during the active production of sweat. The role of thermal (cold) afferents appears to be of a primary importance in driving the perception of skin wetness during the contact with an external stimulus. However, when thermal cues (e.g. evaporative cooling) are limited, individuals seem to rely more on tactile cues (i.e. stickiness and skin friction) to characterise their perception of skin wetness. The central integration of conscious coldness and mechanosensation, as sub-served by peripheral cutaneous A-nerve fibers, seems therefore the primary neural process underpinning humans ability to sense wetness. Interestingly, these mechanisms (i.e. integration of thermal and tactile sensory cues) appear to be remarkably consistent regardless of the modality for which skin wetness is experienced, i.e. whether due to passive contact with a wet stimulus or due to active production of sweat. The novelty of the findings included in this Thesis is that, for the first time, mechanistic evidence has been provided for the neurophysiological processes which underpin humans ability to sense wetness on their skin. Based on these findings, the first neurophysiological sensory model for human skin wetness perception has been developed. This model helps explain humans remarkable ability to sense warm, neutral and cold skin wetness.

<p>From insects to mammals, the ability to constantly sense environmental stimuli is essential for the survival of most living organisms. Most animals have nocifensive behaviors towards extreme temperatures, mechanical stimuli or irritant chemicals that are considered to be noxious. Nociception is defined as the neural encoding and processing of noxious stimuli. This process starts from the activation of pain detecting peripheral sensory neurons (nociceptors) that can detect noxious mechanical, thermal or chemical stimuli. On the other hand, animals also have the ability to discriminate innocuous temperatures and to direct their locomotions to their favorable environmental temperatures and this behavior is called thermotaxis. </p><p>In this study, I used <italic>Drosophila melanogaster</italic>as a genetic model organism to study the molecular and cellular basis of nociception and thermotaxis. <italic>Drosophila</italic> larvae exhibit a stereotyped defensive behavior in response to nociceptive stimuli (termed nocifensive escape locomotion behavior, NEL). Using this behavior as a readout, we manipulated the neuronal activities of periphery sensory Type II multidendritic neurons and have identified a specific class of neurons, class IV multidendritic neurons, to function as nociceptors in <italic>Drosophila</italic> larvae. </p><p>After identifying the nociceptors, I next investigated several ion channels that are critical molecular components for larval nociception. The Degenerin Epithelial Sodium Channel (DEG/ENaC) protein called pickpocket (ppk) is required specifically for larval mechanical nociception but not for thermal nociception. Being specifically expressed in class IV multidendritic neurons (the nociceptors), pickpocket is likely to function as a first detector of mechanical stimuli and upstream of general neuronal action potential propagation. In addition, I have found that the <italic>Drosophila</italic> orthologue of mammalian TRPA1 gene, <italic>TrpA1</italic>, is required for both mechanical and thermal nociception in <italic>Drosophila</italic> larvae. I have cloned a new isoform of dTRPA1 and have found it to be specifically expressed in class IV md neurons. Unlike the known dTRPA1 isoform that is warmth activated, this new isoform is not directly activated by temperatures between 15-42 °C. Instead, it may function downstream of sensory transduction step in the nociceptors. </p><p>Interestingly, <italic>dTrpA1</italic> mutants are also defective in their thermotaxis behavior within innocuous temperature ranges. In addition to the previously reported defects in avoiding warm temperatures, I have found these flies also failed to avoid cool temperatures between 16-19.5 °C. This defect is likely to be mediated by temperature sensing neurons in the antennae. I have detected antennal expression using a GAL4 reporter of dTrpA1. Significantly, these neurons exhibit elevated calcium levels in response to cooling. dTrpA1 mutants have a premature decay of the cooling response at temperatures below 22 °C during a cooling process. I have also identified another population of cells in the antennae that can respond to temperature changes. These neurons express the olfactory co-receptor Or83b and are known to be olfactory neurons. Calcium oscillations triggered by cooling were detected in these neurons and they were terminated by warming. Severe behavioral defects in avoiding cool temperatures were found in animals lacking <italic>Or83b</italic>. Our results suggest that there are multiple pathways regulating cooling sensation in the fly antennae.</p><p>Taken together, I have shown that <italic>Drosophila</italic> serves as a great model system to study nociception and thermosensation at molecular, cellular and behavioral levels.</p> / Dissertation

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